Bioconjugation of Calcium Phosphosilicate Nanoparticles For Selective Targeting of Cells in Vivo

ABSTRACT

Non-aggregating resorbable calcium phosphosilicate nanoparticles (CPNPs) are bioconjugated to targeting molecules that are specific for particular cells. The CPNPs are stable particles at normal physiological pH. Chemotherapy and imaging agents may be integrally formed with the CPNPs so that they are compartmentalized within the CPNPs. In this manner, the agents are protected from interaction with the environment at normal physiological pH. However, once the CPNPs have been taken up, at intracellular pH, the CPNPs dissolve releasing the agent. Thus, chemotherapeutic or imaging agents are delivered to specific cells and permit the treatment and/or imaging of those cells. Use of the bioconjugated CPNPs both limits the amount of systemic exposure to the agent and delivers a higher concentration of the agent to the cell. The methods and principals of bioconjugating CPNPs are taught by examples of bioconjugation of targeting molecules for breast cancer, pancreatic cancer, and leukemia.

Benefit of U.S. Provisional Application No. 61/258,664 filed on Nov. 6,2009 is hereby claimed.

BACKGROUND

Field of the Invention

The present invention relates to the formation and use of bioresorbablecalcium phosphosilicate nanoparticles bioconjugated to molecules thatselectively target cells in vitro and in vivo.

Description of the Art

The early diagnosis of cancer is the critical element in successfultreatment and long term favorable patient prognosis. The high mortalityrate, in particular, for pancreatic cancer is primarily attributed tothe tendency for late diagnoses as symptoms typically occur after thedisease has metastasized as well as the lack of effective systemictherapies. For breast cancer, late diagnosis is often associated withthe lack of timely sensitive imaging modalities. The promise ofnanotechnology is presently limited by inability to simultaneously seek,treat, and image cancerous lesions.

Despite many new advances in the arsenal of antineoplastic agents, drugresistant, highly metastatic cancers continue to ravage patients²¹. Asexamples, breast cancer is still the second leading cause of death inAmerican women with an estimated 192,370 cases diagnosed in 2009. Inthis year alone, about 40,610 women will die from breast cancer in theUnited States. Pancreatic cancer is the fourth leading cause of cancerrelated deaths in the United States. Approximately 42,470 Americans werediagnosed with pancreatic cancer in the past year, and nearly 100% willsuccumb to this disease²¹. It is clear that new modalities must bedeveloped that have the capabilities to both improve diagnosis andtreatment of cancers. The term “theranostic” has been coined to describemodalities that can simultaneously diagnose and treat.

As described in U.S. patent applications Ser. Nos. 10/835,520 and11/142,913, calcium phosphosilicate nanoparticles (CPNPs) have beenengineered to be a resorbable non-toxic vehicle for the delivery of adiversity of therapeutic and imaging agents in biological systems¹⁻⁴.Previous studies have shown that encapsulation within CPNPs improved thelifetime and quantum properties of fluorescent dyes^(1,4). Initial invivo imaging trials demonstrated that CPNPs, functionalized withpolyethylene glycol (PEG) moieties, accumulated within solid tumors viaan enhanced permeation retention (EPR) effect². While EPR serves as aneffective passive targeting strategy, particular interest lies in theability to actively target cancerous cells to deliver anti-neoplasticagents, thereby decreasing effective dosage and limiting off-targettoxicity.

CPNPs are nontoxic, colloidally stable, resorbable, non-aggregatednanoscale vehicles that deliver chemotherapeutics, gene therapy, andimaging agents. Two exciting aspects of CPNPs as drug delivery vehiclesinclude enterohepatic biliary excretion that minimizes hepatic toxicityand pH-triggered release of active agents. At pH 7.4, the CPNPs aresparingly soluble, but the CPNPs dissolve in the late stageendolysosomes at pH 4 to 5^(1,4). The pH response of CPNPs has twodistinct advantages. First, it permits a decrease in the effective doseof chemotherapeutic drugs, which are often toxic, required for optimaltherapeutic benefit by increasing the efficiency of drug delivery intocancer cells³. Second, sequestering the drug in the CPNPs decreases theeffective concentration of free drug present in the extracellular fluidwhere the pH is maintained at approximately 7.4 by physiologicalbuffers. This compartmentalization feature for drug delivery is adistinct advantage since acute systemic toxicity to normal cells islimited. Moreover, off site cytotoxicity may be further ameliorated withtarget and tissue-specific CPNPs.

Scientific investigations have identified cancer cell specific markerswith unique phenotypes that can be exploited to target tumors as will bedescribed in this patent document. Of particular interest is theprevalence of transferrin receptors (CD71) on cancerous cells, includingbreast cancer⁵⁻⁹. The transferrin receptor is responsible fortransporting iron, via interaction with transferrin, into cells asdemanded by metabolic need^(5,6) Accordingly, transferrin receptors arefound predominately on proliferating cells with elevated metaboliclevels, including many cancerous cells, as well as brain capillaryendothelial cells, and hematopoietic cells^(10,11). In a manner similarto CD71, gastrin receptors have a predominate prevalence within certaintissues, specifically the gastrointestinal and central nervoussystems¹²⁻¹⁴. The hormone gastrin binds to a family of G-protein-coupledreceptors, also known as the cholecystokinin-2 (CCK₂ or CCK-B) receptorfamily^(14,15), and is typically known as a key mediator of stomachacidity¹⁶ and growth of the gastrointestinal tract¹⁷. Intriguingly, CCK₂receptor expression is often increased in many cases of gastrointestinalcancer^(14,18) including pancreatic cancer¹⁹ and, in particular, anincrease in expression of a specific splice variant (CCK_(2i4sv) orCCK-C) of the receptor²⁰.

The inventive bioconjugated particles and bioconjugation approachestaught in this patent document may also be used with non-solid tumors.Leukemia is one of the most common and aggressive adult cancers as wellas the most prevalent childhood cancer. Leukemia stem cells (LSCs) havebeen hypothesized to be responsible for cancer development, relapse, andresistance to treatment, and new therapeutics targeting these cellularpopulations are urgently needed. Recently, studies have indicated thatLSCs reside within a lineage ⁻Sca-1⁺ CD117 cellular population in humanpatients and animal models of chronic myeloid leukemia (CML) andtherefore present a target for intervention.

Accordingly, as outlined above, there is a significant medical need inthe field of disease treatment for nanoparticle compositions capable oftargeted systemic delivery of imaging and/or therapeutic agents as wellas imaging and treatment methods employing such nanoparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are shown in thedrawings exemplary embodiments of the invention; however, the inventionis not limited to the specific methods, compositions, and devicesdisclosed. In addition, the drawings are not necessarily drawn to scale.

FIGS. 1A-1C schematically outline non-covalent and covalent methods forbioconjugating targeting molecular moieties to CPNPs.

FIG. 2 illustrates Zeta potential distributions for citrate-CPNPs,Avidin-CPNPs, PEG-CPNPs, and gastrin-10-PEG-CPNPs. The citrate-CPNPsdisplayed a mean zeta potential of −16±1.3 mV, whereas PEGylationshifted the mean zeta potential to +3.0±2.0 my, gastrin-10 conjugationfurther shifted the mean zeta potential to +6±3.2 mv, and theavidin-CPNPs had a mean zeta potential value of +29±8.7 mV. All zetapotential distributions are representative of three independentexperiments.

FIG. 3 shows dynamic light scattering determinations for citrate-CPNPs,anti-CD71-Avidin-CPNPs (Anti-CD71-AV-CPNP), HumanHolotransferrin-Avidin-CPNPs (Tf-Av-CPNP), Pentagastrin-Avidin-CPNPs(PG-Av-CPNP), maleimidePEG-CPNPs (Peg-CPNP), andgastrin-10-maleimidePEG-CPNPs (gastrin-10-PEG-CPNP). All dynamic lightscattering determinations are the mean of three independent experiments.Inset shows a typical TEM micrograph of Citrate-CPNPs.

FIGS. 4A-4D illustrate the binding and displacement of 2,6-ANS utilizedto evaluate the coupling of biotin to avidin-CPNPs. FIG. 4A showsfluorescence intensities for the first step of the 2,6-ANS assay. Theaddition of 2,6-ANS to the avidin-CPNP complex results in a six foldincrease in fluorescence as the fluorescent probe binds to the biotinbinding site on avidin. The 2,6-ANS was added at increasingconcentrations to avidin-CPNPs and increased fluorescence, indicative of2,6-ANS bound to avidin, was quantitatively determined. FIG. 4B showspeak height of fluorescence shown on FIG. 4A as a function of 2,6-ANSmolarity. FIG. 4C shows fluorescence intensities for the second step ofthe 2,6-ANS assay. The addition of biotin to the 2,6-ANS-Avidin-CPNPcomplex results in a decrease in fluorescence as biotin displaces thefluorescent probe from the biotin binding site on avidin. Biotin wasadded at increasing concentrations to the 2,6-ANS-Avidin-CPNP complexand a decrease in fluorescence, indicative of biotin displacing 2,6-ANS,was quantitatively determined. FIG. 4D shows peak height of fluorescenceshown on FIG. 4C as a function of biotin molarity. All determinationsare representative of three independent experiments.

FIGS. 5A-5C illustrate targeting transferrin receptors in an in vivosubcutaneous-tumor model of breast cancer. Human MDA-MB-231 metastaticbreast cancer cells were xenografted subcutaneously into athymic nudemice. One week following engraftment, ICG-loaded CPNPs were administeredsystemically via tail vein injection and near-infrared images were taken24 hours post-injection. FIG. 5A shows flow cytometry demonstrating thepresence of the transferrin CD71 receptor. FIG. 5B shows near-infraredimages taken 96 h post injection. From left to right, mice received: (i)free ICG, (ii) ICG-loaded, PEG-CPNPs, (iii) ICG-loaded,anti-CD71-Avidin-CPNPs, and (iv) ICG-loaded, HumanHolotransferrin-Avidin-CPNPs. 5C: Excised tumors (mice ii, iii, and iv),excised spleen (mouse iii), and excised stomach (mouse iii). All imagesare representative of four independent experiments.

FIG. 6 illustrates ICG-loaded PEG-CPNP clearance via hepatobiliarysecretion. Twenty-four hours post-tail vein injection, the kidney,liver, spleen, and intestine were excised and imaged. Increased signalis seen toward the end of the intestine as indicated by fecal pelletswithin the intestine. All images are representative of three independentexperiments.

FIGS. 7A-7B illustrate that gastrin bioconjugated CCK₂-receptor-targetedCPNPs effectively targeted human BxPC-3 pancreatic cancer cells. BxPC-3cells were exposed to flourescein-loaded untargeted PEG-CPNPs orGastrin-10-PEG-CPNPs, for 5 min. followed by exchange to fresh media for55 min, or exposure for 60 min. FIG. 7A shows cells fixed and visualizedby microscopy. FIG. 7B shows cells fixed and analyzed by flow cytometrywith graphs representing 10,000 collected events per sample.

FIGS. 8A-8B illustrate targeting gastrin receptors in an in vivoorthotopic-tumor model of pancreatic cancer. Human BXPC-3 pancreaticcancer cells were xenografted orthotopically into athymic nude mice.FIG. 8A shows near-infrared images. One week following engraftment,ICG-loaded CPNPs were administered systemically via tail vein injectionand near-infrared images were taken 96 hours post-injection. From leftto right, mice receiving: (i) ICG-loaded, PEG-CPNPs, (ii) ICG-loaded,Gastrin-10-PEG-CPNPs (covalently coupled), or (iii) ICG-loaded,Pentagastrin-Avidin-CPNPs. FIG. 8B shows excised, tumor-bearing pancreasfrom each mouse and excised brain (mouse ii). All images arerepresentative of at least four independent experiments.

FIG. 9 illustrates the efficacy of targeted ICG-CPNP PDT in vitro.32D-p210-GFP chronic myeloid leukemia cells were treated with PBS, empty(ghost)-CPNPs, ICG-loaded CPNPs, or CD117-targeted ICG-loaded CSNPsfollowed by NIR-laser treatment. Cells from a human acute myeloidleukemia patient (#202) were treated with PBS, empty (ghost)-CPSNPs,ICG-loaded CPSNPs, or CD96-targeted ICG-loaded CPSNPs followed byNIR-laser treatment. CD96-targeted ICG CPSNP PDT was evaluated in asimilar manner with cells from another human acute myeloid leukemiapatient (#370).

FIG. 10 illustrates that the leukemia burden is reduced byCD117-targeted ICG-CPNP PDT in vivo. Leukemia was established in C3H/HeJmice with 32D-p210-GFP cells, and mice were treated with PBS, empty(ghost)-CPNPs, ICG-loaded CPNPs, or CD117-targeted ICG-loaded CPNPsfollowed by NIR-laser treatment. The leukemia burden was followed byroutine flow cytometry analysis of GFP+ leukemic cells in the blood.Representative flow cytometry dot plots are shown.

FIG. 11 illustrates that survival is extended by CD117-targeted ICG-CPNPPDT in vivo. Leukemia was established in C3H/HeJ mice with 32D-p210-GFPcells, and mice were treated with PBS, empty (ghost)-CPNPs, ICG-loadedCPNPs, or CD117-targeted ICG-loaded CPNPs followed by NIR-lasertreatment and survival was monitored.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to thespecific devices, methods, applications, conditions or parametersdescribed and/or shown herein, and that the terminology used herein isfor the purpose of describing particular embodiments by way of exampleonly and is not intended to be limiting of the claimed invention. It isto be appreciated that certain features of the invention, which are, forclarity, described herein in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention that are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any subcombination.

This patent document describes the design and synthesis of resorbablebioconjugated calcium phosphosilicate nanocomposite particles (CPNPs)that can be systemically targeted to biological tissues by theattachment of appropriate molecular moieties that specifically recognizeand bind to the target tissues. The general methodology for synthesizingand using these bioconjugated CPNPs is disclosed through three examplebioconjugation techniques. Using these techniques, fluorescently labeledCPNPs are described that 1): systemically target breast cells; 2)systemically target pancreatic cancer lesions; and 3) systemicallytarget leukemia stem cells. A fluorescent label incorporated into theCPNPs permits identification of the location of binding of the CPNPs inbreast and pancreatic cancers, and, in the case of leukemia stem cells,the photodynamic destruction of the cells. As taught in the prior art³other agents including chemotherapeutic agents can be incorporated intothe CPNPs in place of or alongside ICG. Thus, the bioconjugated CPNPstaught in this patent document can be utilized both for identificationof the presence and location of diseased tissue as well as the treatmentof diseased tissue by the targeted delivery of therapeutic agents.

The bioconjugated CPNPs used for purpose of disclosure in this patentdocument comprise a ˜20 nm diameter composite particle composed of anamorphous calcium phosphate matrix doped with silicate in which a nearinfra-red imaging agent, indocyanine green (ICG) is embedded. Thefollowing three coupling strategies for bioconjugation to CPNPs aredescribed and their use is disclosed herein in the following exemplaryembodiments:

-   -   CPNP-Avidin non-covalently bonded to biotinylated anti-CD71    -   CPNP-Avidin non-covalently bonded to biotinylated human        holotransferrin ligand    -   CPNP-Avidin non-covalently bonded to biotinylated pentagastrin    -   CPNP-PEG (maleimide-coupled) covalently bonded to gastrin-10    -   CPNP-PEG (sulfo-NHS-coupled) covalently bonded to anti-CD117    -   CPNP-PEG (sulfo-NHS-coupled) covalently bonded to anti-CD96

Example 1—Avidin Biotin Coupling

The first coupling strategy schematically outlined in FIG. 1A employs anavidin molecule coupled to citrate functionalized CPNPs. (A variety ofcarboxylate surface functionalizations may be used in the preparation ofthe CPNPs. Citrates are considered particularly suitable sources ofcarboxylate surface functionalization. In some embodiments, the surfacefunctionalization (e.g., citrate) is specifically adsorbed to thecalcium phosphosilicate nanoparticle. Avidin has four potential bindingsites for biotin. As is well known in the art, many biotinylatedbiological molecules of interest are available that can be used totarget specific cell types. An avidin-CPNP is described that can be usedto non-covalently bioconjugate targeting biological molecules to theCPNPs by coupling the biotinylated targeting molecule of interest toavidin-CPNPs. In this patent document three working examples describeCPNP bioconjugation to human holotransferrin, anti-CD71 antibody, and ashort gastrin polypeptide (pentagastrin) by means of avidin-biotincoupling strategies. The conjugation of biotinylated humanholotransferrin (diferric transferrin), biotinylated anti-CD71 antibody(antitransferrin receptor antibody), and biotinylated pentagastrin toavidin conjugated CPNPs (avidin-CPNPs) permited the attempted targetingof transferrin receptors, which are highly expressed on breast cancercells. In a similar manner, the conjugation of biotinylated pentagastrinto avidin-CPNPs permited the attempted targeting of CCK₂ receptors.

Example 2—Maleimide Covalent Coupling

A second covalent coupling strategy schematically outlined in FIG. 1Buses a PEG-maleimide reaction to bioconjugate a targeting molecule andis exemplified by the coupling of decagastrin (gastrin-10). Targetingmolecular moieties suitable for use with this method include thosehaving a sulfhydryl-group such as antibodies, peptides, ligands,receptors, and the like. Citrate functionalized CPNPs are reacted with acarbodiimide and a maleimide polyethylene glycol amine to yield a CPNPcomprising a surface moiety having a maleimide terminal group that canthen be reacted with a binding moiety having a sulfhydryl group.Ethyl-N-(3-dimethylaminopropyl)-N′ hydrochloride carbodiimide isconsidered especially suitable for the disclosed method for thecarbodiimide linker group. The maleimide group is suitably bound to thesurface of the nanoparticle, but can, in some embodiments, be bound tothe binding moiety. PEG-maleimide coupling of decagastrin (gastrin-10)to PEG-CPNPs also permits targeting of gastrin receptors, which areover-expressed in pancreatic cancer lesions, but not normal pancreas.Maleimide coupling of gastrin to a targeting peptide is described in theprior art³⁵, but the present invention of using maleimide chemistry tocouple a targeting moiety to a resorbable non-aggregating CPNPincorporating an imaging or therapeutic agent is not taught oranticipated by the prior art.

Example 3—Covalent Coupling via N-Hydroxysulfosuccinimide (Sulfo NETS)

The bioconjugation of anti-CD96 and anti-CD117 antibodies and othertargeting moieties not possessing a thiol group requires a differentapproach since the maleimide-coupling approach relies on theavailability of thiol groups on the molecule to be conjugated, which ifunavailable, necessitates thiol introduction under conditions that mayjeopardize the integrity of the biomolecules. To avoid this potentialproblem and provide a generalized coupling method that does not rely onthe availability of thiol groups, a third coupling strategy outlined inFIG. 1C was developed that utilizes a multistep synthesis using EDAC andSulfo NHS.

As will be appreciated by those skilled in the art, targeting molecularmoieties suitable for bioconjugation may include antibodies,polypeptides, nucleic acids such as siRNAs, ligands, receptors and thosemoieties that target signaling proteins, angiogenesis factors,metalloproteases, and the like. Generally, the targeting moieties may bechosen on the basis of their ability to bind specifically to aparticular tissue such as a cancerous cell and/or cancerous lesion.

All these bioconjugated CPNPs have the potential to perform as atheranostic modality, simultaneously enhancing drug delivery, targeting,and imaging of breast, pancreatic, and leukemic cancers.

Physical Characterization of CPNPs:

Citrate functionalized CPNPs were utilized as a platform for avidination(see experimental procedures below for description of bioconjugationschemes such as avidination) which allowed the characterization ofbioconjugation via zeta potential analysis. FIG. 2 shows the zetapotential distribution of: 1) citrate-CPNPs prior to bioconjugation withavidin; 2) of the avidin-CPNPs complex; 3) maleimide-terminatedpolyethylene glycol (PEG)-coated CPNPs; and 4) CPNPs conjugated withgastrin-10 via a maleimide-PEG coupling . Prior to bioconjugation withavidin, the Citrate-CPNPs display a negative mean zeta potential valueof −16±1.3 mv, which is consistent with previous reports¹. However,after bioconjugation with avidin, the Avidin-CPNPs displayed arelatively high positive mean zeta potential value of +29±8.7 mv. Theisoelectric point for avidin is pH 10. Thus, the shift from a negativezeta potential to a positive zeta potential distribution is strongevidence of avidin bioconjugation on the surface of CPNPs. Also, FIG. 2shows the shift in mean zeta potential to +3.0±2.0 my when coated withthe PEG and then a further shift to +6±3.2 mv when conjugated togastrin-10.

Further characterization confirmed the presence and bioactivity of thebioconjugated Avidin-CPNPs for biotin. A 2,6-ANS titration was used toconfirm both the presence of avidin and it associated bioactivity. Ananalysis of the particle size distributions of the nanoparticles bymeans of dynamic light scattering (DLS) as shown in FIG. 3 revealed thatall the various bioconjugated CPNPs had a larger mean hydrodynamicdiameter that the non-bioconjugated citrate-CPNPs. Additionalcharacterization was performed using transmission electron microscopy(TEM). Transmission electron microscopy analysis indicated that theinorganic particle size was in the range from 10 to 30 nm for all of theCPNPs (see inset in FIG. 3). The smaller size indicated by TEM relativeto DLS analyses is consistent with the ability to determine the solidmaterial diameter by the TEM technique in contrast to DLS which givesthe hydrodynamic size distribution in colloidal suspension of solidparticle, organic layers, and surrounding liquid.

Previous studies have demonstrated that a 2,6-ANS assay can be utilizedto evaluate the biotin-binding functionality of avidin²². The 2,6-ANSfluorescent probe binds to avidin, an event that can be measured usingfluorescence spectroscopy. Without avidin present, the 2,6-ANSfluorescent probe displays low fluorescence intensity. In the presenceof avidin, the binding of 2,6-ANS to the biotin-binding site on avidinproduces an increase in fluorescence intensity. 2,6-ANS added inincreasing concentrations to the avidin-CPNPs yielded aconcentration-dependent increase in 2,6-ANS fluorescence emission (seeFIGS. 4A and 4B). The titration of 2,6-ANS showed an increase influorescence up to 1.79×10⁶ RFU after the addition of 34 μM 2,6-ANS.Beyond this point of maximum fluorescence intensity, 2,6-ANSself-quenched, at which point biotin was added to displace the 2,6-ANS.Biotin has a greater affinity for the biotin-binding site on avidin thandoes the 2,6-ANS fluorescent probe. Therefore, biotin additions to the2,6-ANS-Avidin-CPNP complex displaces the 2,6-ANS from thebiotin-binding site on avidin. Since the 2,6-ANS fluorescent probedisplays minimal fluorescence when it is not bound to avidin, thisdisplacement produces a decrease in fluorescence (FIGS. 4C and 4D) to aplateau of 1.08×10⁶ RFU after the addition of 1.90 nM biotin. Theplateau is present in FIG. 4D because of the intrinsic fluorescence of2,6-ANS. This result demonstrates the successful coupling of biotin tothe Avidin-CPNPs. The 2,6-ANS fluorescence emission did not decreasecompletely as some 2,6-ANS remains bound to the Avidin-CPNPs. While theaffinity of avidin for biotin is high, residual reactants and ionicconditions can influence this affinity as it has been established thatwater participates in displacing biotin from the binding pocket ofavidin, or similar proteins23,24. Nonetheless, this analysissuccessfully demonstrates that the Avidin-CPNPs are biofunctional,through binding of 2,6-ANS as well as its displacement by biotin. One ormore of the four avidin binding sites for biotin may be occupied when abiotinylated targeting molecule is bound to avidin, the exact numberdepending on the location of the avidin on the CPNP and the orientationand size of the biotinylated molecule.

Evaluation of Breast Cancer-Targeted CPNPs In Vivo:

Transferrin receptors are expressed on cells with increased metabolicdemand, including several cancerous cells. The presence of thetransferrin receptor (CD71) on the surface of human MDA-MB-231 cells wasdetermined via flow cytometry and was found to be prevalent on nearlyall cells analyzed (FIG. 5A). The presence of CD71 on most MDA-MB-231cells indicated that it would be an ideal surface target, exploited bycoupling specific antibodies, or the ligand holotransferrin, todemonstrate the utility of bioconjugated Avidin-CPNPs. It has beenpreviously shown that the untargeted PEG-CPNPs passively accumulate inbreast cancer tumors via the EPR effect². This finding was successfullyrepeated by the present inventors as a positive control (FIG. 5B).Intriguingly, only tumors from mice receiving anti-CD71-Avidin-CPNPs,and not Human Holotransferrin-Avidin-CPNPs or untargeted PEG-CPNPs, wereeffectively targeted as evidenced by prominent infrared stimulatedflorescence of ICG at 96 h following tail vein injection of CPNPs (FIG.5B). It has been reported, and is likely in this circumstance, that thetransferrin receptors are saturated with transferrin²⁵ and therefore areunable to bind the Human Holotransferrin-Avidin-CPNPs. This is alsosupported by the success of the anti-CD71-Avidin-CPNPs, which recognizean epitope separate from the ligand-binding site on the transferrinreceptor. Importantly, the anti-CD71-Avidin-CPNPs were more effective attargeting the tumors than the passively accumulating PEG-CPNPs based onthe relative fluorescence intensity. However, and not surprisingly, theeffective targeting was not limited to the tumors, but also to thespleen, which is rich in a diversity of hematopoietic cells (FIG. 5C).It was also observed that there was some off-target staining of thestomach (FIG. 5C), possibly due to avidin interaction with biotiningested as part of the mouse's diet or due to the presence oftransferrin receptors on these tissues. Previously, clearance ofPEG-CPNPs was reported to occur via hepatobiliary clearance evidenced bypredominant staining of the liver within minutes following tail veininjection. In these experiments, hepatobiliary clearance was validated24 hours post-tail vein injection of PEG-CPNPs, and showed theprogression of signal from the liver and through the intestine as fecalmatter (FIG. 6). Overall, it has been demonstrated that the transferrinreceptor-targeted CPNPs were effective and selective in imagingcancerous tissues in an in vivo model of breast cancer.

Evaluation of BxPC-3 Pancreatic Cell Targeting by Gastrin-10 ConjugatedCCK₂ Receptor-Targeted CPNPs In Vitro:

Increased surface expression of CCK₂ receptors on pancreatic tumors, andcell lines, was targeted by CPNPs coupled via a PEG maleimide linker toa short gastrin peptide (gastrin-10-PEG-CPNPs). (This moiety was chosenbecause it is shorter than the biologically active form, gastrin-17, butpreserves targeting specificity) BxPC-3 human pancreatic cancer cellswere treated with gastrin-10-PEG-CPNPs or untargeted PEG-CPNPs for 5 or60 minutes, followed by a replacement of media for 55 minutes or nochange, respectively. Cells were fixed and visualized using afluorescence microscope set up to analyze a broad range of fluorescencesimultaneously. Only BxPC-3 cells exposed for 60 minutes togastrin-10-PEG-CPNPs, and no media exchange, displayed fluorescentstaining (FIG. 7A). The observed fluorescence was green and blue,indicative of the pH-dependent degradation of CPNPs as they internalizein the endosomal-lysosomal pathway, and release the encapsulated dye(fluorescein). Fluorescein displays a complex pH-dependent equilibriumand emission from its two fluorescent ionic forms, the monoanion anddianion.^(26,27) In higher pH environments, such as that within theCPNPs and physiological solutions, the significant emission wavelengthis from the dianion (peak excitation 495 nm, green). As pH drops below6.5, the molecule is protonated to its monoanionic form which is excitedin the blue (450 nm). Thus, emission signals from thefluorescein-encapsulating CPNPs shift from green toward blue asfluorescein molecules experience the pH drop characteristic of theendosomal-lysosomal pathway into the cells, resulting in the dissolutionof the particles and release of the fluorophore into the lower pHenvironment of late stage endosomes.

Alternatively, BxPC-3 cells were exposed, fixed, and analyzed via flowcytometry (FIG. 7B). This further showed that gastrin-10-PEG-CPNPs (60minutes exposure) targeted BxPC-3 cells while untargeted PEG-CPNPs didnot.

Evaluation of Pancreatic Cancer-Targeted CPNPs In Vivo:

Evaluation of pancreatic cancer-targeted CPNPs in vivo indicates thatthe untargeted, PEG-CPNPs effectively accumulated by EPR 24 h post-tailvein injection within small BXPC-3 tumors within the pancreas (FIG. 8A),and these whole animal images were confirmed by excision of the pancreas(FIG. 8B). Pentagastrin-Avidin-CPNPs (iii), using the avidin-biotincoupling approach, also targeted the pancreatic tumors (FIG. 8).However, the gastrin-10-PEG-CPNPs (ii) proved to be much more successfulat targeting the pancreatic tumors (FIG. 8A), including peritonealextensions of the primary tumor, as well as the brain, which is alsorich in CCK₂ receptors¹⁴. Targeting of the gastrin-10-PEG-CPNPs to thebrain was confirmed by excising and imaging the brain during necropsy(FIG. 8B).

An advantage of the later targeting approach is the covalent attachmentof the targeting moiety (gastrin-10), eliminating the possibility ofnon-specific avidin interactions in vivo, as well as the PEG, whichpermits improved systemic retention and decreased immune-reactivity². Itis also possible that the presence of avidin on the CPNPs does notpermit crossing of the blood-brain barrier, whereas the PEG-maleimidebioconjugation for gastrin-10 may permit penetration of the blood-brainbarrier. The challenge in the prior art of delivering a blood-bornetherapeutic to the brain across the blood brain barrier is well known.The present invention overcomes this problem and is one of the mostimportant aspects of the invention. It is noteworthy that the untargetedPEG-CPNPs did not display any significant brain accumulation in thisstudy. A recent work comparing interleukin-13-targeted nanoliposomes tountargeted nanoliposomes in a cranial model of glioblastoma showed thatonly targeted nanoliposomes moved significantly across theblood-brain-barrier²⁸. The results presented in this patent document,although using a different target and different nanoparticles (CCK₂receptor-targeted CPNPs), corroborates the findings by others thattargeted nanoparticles can cross the blood-brain-barrier²⁵. Importantly,the CPNPs are biocompatible, and it has been previously shown that theyexhibited no specific detrimental effects toward neurons³. Thisimportant result was confirmed in these experiments, as no micereceiving any CPNPs showed signs of neurological deficits. Overall, theinventive bio-conjugated CPNPs and their method of use demonstrate thatthe CPNPs can be effectively targeted to CCK₂ receptors in vivo in amodel of pancreatic cancer, and further demonstrate the potential fortargeting across the blood-brain-barrier.

Targeting of Leukemia Stem Cell for Photodynamic Therapy:

Photodynamic therapy (PDT) has been described as an alternative tochemotherapy or radiation therapy in the treatment of malignant tumors⁶.PDT consists of three components: a photosensitizer, light, and oxygen.When exposed to light of a specific wavelength, a photosensitizer isexcited, and a subsequent energy transfer to molecular oxygen producessinglet oxygen. Highly reactive singlet oxygen rapidly reacts withnearby cellular components, ultimately leading to cell death. As notedearlier, ICG may be encapsulated in CPNPs and currently is FDA approvedas a contrast agent and works as a PDT agent upon illumination. CD117, areceptor tyrosine kinase, also known as c-kit, is the receptor for stemcell factor (SCF), and is normally internalized by ligand binding. CD117internalization is dependent on tyrosine kinase activity, and thisprocess has been shown to be important to the pro-growth signalingmechanisms elicited by SCF. ICG-loaded CPNPs were bioconjugated toanti-CD117 antibodies targeting surface features expressed on LSCs. Flowcytometry, was used to verify the ability of anti-CD117antibody-coupled-PEG CPNPs to target cells of interest. As also notedearlier, CPNPs internalized into the endosomal-lysosomal pathway degradeand release ICG.

Targeting In Vitro:

In an in vitro model, 32D-p210-GFP murine CML cells, and human AMLpatient cells, were evaluated for sensitivity to PDT utilizingICG-loaded CPNPs. (FIG. 9) Not surprisingly, PDT using non-targetedCPNPs loaded with ICG had only minimal, yet significant, efficacy in32D-p210-GFP cells (30% reduction in viability), and absolutely noefficacy in both AML patient samples evaluated. In contrast, PDTutilizing anti-CD117-targeted CPNPs loaded with ICG exerted a profoundand significant anti-leukemic effect on 32D-p210-GFP cells (51%reduction in viability), an effect that was also significantly differentfrom that observed with untargeted CPNPs loaded with ICG. A robust andsignificant anti-leukemic effect was observed with PDT of two AMLpatient samples utilizing CD96-targeted CPSNPs loaded with ICG (40% and33% reductions in viability of samples). Altogether, these results showthat targeted therapy, and in particular LSC-targeted therapy utilizingbio-conjugated ICG-loaded CPNPs can improve the efficacy of PDT.

Targeting In Vivo:

Anti-CD117-PEG-ICG-CPNPs were employed in an in vivo murine model ofCML. CPSNPs were diluted approximately 1:10 into PBS (200 nMpre-injection concentration of ICG), or controls, injected systemicallyinto the lateral tail vein, and were followed immediately by 12.5 J/cm²laser NIR irradiation of the spleen. PDT utilizing non-targeted CPNPsloaded with ICG effectively slowed leukemia progression in vivo asevidenced by a significantly slower growth of 32D-p210-GFP cells in themurine model of CML (FIG. 10). This translated to a significant increasein the lifespan of these animals (FIG. 11). These results demonstratedthat PDT using ICG-loaded CPNPs was efficacious in an in vivo model ofCML, despite an inability of the untargeted CPNPs to accumulate viaenhanced permeability and retention. Most importantly, PDT utilizingLSC-targeted CPSNPs loaded with ICG evoked a robust anti-leukemiceffect. In particular, anti-CD117-targeted CPNPs loaded with ICG afterPDT illumination effectively blocked an increase in leukemic cells inthe blood (FIG. 10). This blockade of leukemic cell proliferation invivo correlated with a profound and dramatic extension in survival (FIG.11) including leukemia-free survival.

Biocojugation of Systemically Targeted CPNPs:

The ability to target nanodelivery systems to specific tissues isimportant in the development of improved therapeutics for diseases suchas cancer. In many studies and clinical circumstances, the efficacies oftreatment are limited or the off-target effects are dramatic. Nanoscaletherapeutics help to minimize these problems by concentrating higherdoses of therapeutic agents in the target tissues while reducing theconcentration of therapeutic agents systemically.

In the prior art it has been shown that molecular-specific therapeutics,or targeted therapeutic delivery systems are highly efficacious and mayeven help to overcome complicating circumstances such as multidrugresistance¹¹. Often, prior art studies were restricted to in vitromodels. However, the true test for active targeting requires in vivomodels, in which the delivery modality is administered systemically,with the nanodelivery system allowed to freely circulate to localize inthe desired tissue to establish efficacy of the targeting strategy anddelivery system. This inventions described in this patent documentdemonstrate for the first time the bioconjugation of targeting moietiesto CPNPs to effect systemic targeting in a subcutaneous, an orthotopic,and haemopoetic in vivo model using the CPNP nanodelivery system.

The CPNPs utilized for bioconjugation were engineered specifically asnon-toxic, resorbable, biocompatible nanoscale delivery vehicles. It hasbeen previously shown that a variety of molecules, including dyes thatcould be used in tumor detection and photodynamic therapy, orhydrophobic antineoplastic agents such as ceramide could beencapsulated¹⁻³ in the CPNPs. Until now in the prior art, CPNPs haverelied on passive accumulation via enhanced permeation retention effect,to ‘target’ solid tumors and had not been employed against non-solidtumors. For the first time, the disclosures in this patent documentdemonstrate that surface targeting molecular moieties can besuccessfully bioconjugated to the CPNPs, via synthetically appropriateand distinct coupling methods, and that these bioconjugated CPNPs caneffectively target select tissues and stem cells via surface featuretargeting/recognition. Specifically, breast cancer tumors were targetedin vivo by targeting transferrin receptors, orthotopic pancreatic cancertumors were targeted in vivo by targeting gastrin receptors, andleukemic stem cells were targeted in vivo by CPNPs bearing an anti-CD117antibody (an anti-CD96 antibody in the case of human patient samples invitro).

The observation in the prior art was also confirmed that the untargeted,PEG-CPNPs could accumulate moderately yet could be effectively imagedwithin small orthotopic pancreatic tumors, extending the diagnosticimaging capability and therapeutic delivery capabilities to one of themore evasive cancers. In addition, it is demonstrated thatgastrin-receptor targeted CPNPs could cross the blood-brain barrier,which may expand the utility of the CPNPs to therapeutics targeted toglioblastoma or even to neurodegenerative or psychiatric disorders.

Even though systemically targeted bioconjugated CPNPs can be used totarget-specific receptors or ligands on cancer cells, there may still beobstacles, as these receptors may still be expressed on other tissues.As a case in point to alleviate this short-coming, CPNPs can potentiallybe ‘loaded’ with selective gene therapies or agents to becomecancer-specific. For example, although CCK₂ receptors are present inboth malignant and some normal tissues, only the pancreatic cancer cellsproduce endogenous gastrin²⁹. The acid secreting parietal cells of thestomach, imaged as described in this patent document with ICG-loaded,gastrin-10-PEG-CPNPs (covalently coupled), or ICG-loaded,Pentagastrin-Avidin-CPNPs (non-covalently coupled), do not produceendogenous gastrin. The only nonmalignant cells that produce gastrin inadults are the G-cells of the stomach antrum; and, the G-cells do notpossess CCK₂ receptors^(30,31). Therefore, loading the CPNPs with a genetherapy agent (i.e., siRNA) or a gastrin antagonist will be effective intreating cancer cells without harming noncancerous cells.

It been shown in the prior art that down regulation of endogenousgastrin expression using RNA interference techniques significantlyinhibits growth of pancreatic cancer tumors and metastases^(32,33). Oneproblem with using gene therapy in animals and in humans has been infinding delivery systems that would protect the siRNA from degradationin the peripheral circulation. Since siRNAs are readily degraded bynucleases in the peripheral blood and tissues, mechanisms for deliveryhave been an active area of recent investigation. Viral vectors,especially the adeno-associated viruses (AAVs) and the adenoviruses havebeen under investigation; however, hepatotoxicity and immunogenicityhave been reported³⁴. Based on the teachings in this patent document, atissue-specific and cancer-selective vehicle such as siRNA-loaded CPNPscoupled for receptor targeting can be created and would be ideal ascancer therapeutics. It should be noted that virtually any antibody issuitable for use as a targeting moiety. Similarly, a wide range ofpeptides are suitable for use in the present invention.

Altogether, the successful bioconjugation of selective surface targetingmoieties to CPNPs using a variety of coupling approaches has beendisclosed demonstrating the effectiveness, selectivity, and utility inthree separate in vivo models. The techniques disclosed will allow thefurther development of the CPNPs to target a diversity of disorders,including several poor prognosis cancers.

Experimental Procedures: Preparation of Nanoparticles:

CPNPs were prepared by the microemulsion technique and van derWaals-HPLC that have been previously described¹⁻⁴. Cyclohexane (C₆H₁₂,99%, BHD Chemical Co.), Igepal CO-520 (C₁₃H₂₀O(C₂H₄O)_(n=5), RhodiaChemical Co.), and deionized H₂O were used to prepare themicroemulsions. Calcium chloride (CaCl₂. 2H₂O, Sigma-Aldrich Co.),disodium hydrogen phosphate (Na₂HPO₄, Sigma Aldrich Co.), and sodiummetasilicate (Na₂SiO₃, Sigma-Aldrich Co.) were used as particleprecursors. Disodium hydrogen citrate dihydrate(HOC(COOH)(CH₂COONa)₂.2H₂O, Sigma-Aldrich Co.) was used as thedispersant. Indocyanine green (ICG) (TCI America Co.) was used as thenear infrared fluorophore in the CPNPs for the animal trials, whilefluorescein sodium salt (Sigma-Aldrich Co.) was the visible fluorophoreencapsulated for flow cell and in vitro experiments. Neat ethanol waspurchased from VWR International. All other chemicals were obtained fromSigma-Aldrich Co., unless otherwise noted.

Two separate Microemulsions (1 and 2) were formed with acyclohexane/Igepal CO-520/water system. The molar ratio of water tosurfactant was 4. A 650 μl of 1×10⁻² M CaCl₂ in CO₂-free deionized H₂Owas added to 14 ml of a 29 volume percent solution of Igepal CO-520 incyclohexane to form Microemulsion 1. Similarly, 65 μl of 6×10⁻² Mdisodium hydrogen phosphate (Na₂HPO₄) with 65 μl of 8.2×10⁻³ M sodiummetasilicate (Na₂SiO₃) in CO₂-free deionized H₂O (pH 7.4) was added to14 ml of a 29 volume percent solution of Igepal CO-520 in cyclohexane toform Microemulsion 2. A 520 μl aliquot of 0.01 M ICG in CO₂-freedeionized H₂O was added into Microemulsion 2 so that the final H₂Ovolume matched that in Microemulsion 1 (650 μL), hence retaining thewater to surfactant ratio in each. The individual microemulsions wereallowed to equilibrate for 1 hour before 1 and 2 were mixed to formMicroemulsion 3. Microemulsion 3 was allowed to undergo micellarexchange for 2 minutes, during which time doped CPNPs precipitated inthe micelles. A 225 μl aliquot of 1×10⁻² M sodium citrate was added toMicroemulsion 3 and allowed to react for 15 minutes. After adding thedispersant, the reverse micelles were dissolved with 50 ml of ethanoladjusted with 1 M KOH before laundering via the van der Waals-HPLC¹⁻⁴.).

The unwashed CPNP suspension was loaded onto a silica HPLC (highperformance liquid chromatography) column after the micelles had beendissolved with ethanol; the free organic was laundered with ethanoladjusted with 1 M KOH as the eluent; finally, the particles were elutedusing 70:30 ethanol:water by volume. During the washing step, the dyecontent was monitored at an absorption of 785 nm or 495 nm for ICG orfluorescein respectively. The ethanol washing was continued until thedetector reached baseline indicating removal of theexcess dye. The firstmajor peak was collected. The precursor and HPLC solutions were preparedwith CO₂-free deionized H₂O to avoid carbonate contamination in theCPNPs. All solution pH measurements were performed using a Sentron ISFETpH probe calibrated against aqueous standards.

Bioconjugation of CPNPs:

Avidin Coupling:

To bioconjugate the CPNPs with avidin, a 1 ml aliquot of CPNPs in their70% ethanol solution was first dried under argon and covered from lightuntil all of the solvent evaporated and only theCPNPs remained. Thisdried sample was then reconstituted back to 1 ml with the addition of 1ml PBS (0.01 M phosphate buffer, 0.14 M NaCl, 0.01 M KCl at pH 7.4).This was then followed by the addition of 1 ml of 20 mg/ml1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDCI)(Sigma-Aldrich Co.) and 1 ml of 6 mg/ml avidin (Rockland ImmunochemicalsInc.). Excess avidin was added in order to ensure the surface saturationof CPNPs. This reaction mixture was finally incubated at 35° C. for 24hours in the dark with continuous stirring at 600 rpm.

After 24 hours, the reaction mixture was centrifuged (Marathon 22KCentrifuge; Fisher Scientific Co.), filtered with a 100 kDa centrifugefilter device (Amicon Ultra-4, PLHK Ultracel-PL Membrane; Millipore Co.)to remove excess unconjugated avidin. Prior to use, the filtrationmembrane of the centrifuge filter device was washed with deionized H₂Oin order to minimize non-specific binding. A total of three centrifugefiltrations, each at 1000×g for 30 minutes, were performed in order tomaximize the removal of excess unconjugated avidin. After eachcentrifuge filtration step, the final volume of the retentate solutionwas brought back to the starting volume by the addition of PBS.

Multiple types of commercially available centrifuge filter devices, withdifferent filtration membrane materials and chemistry, were evaluated toobtain minimal non-specific adsorption and optimal washing of the CPNPs.The Millipore Amicon centrifuge filter device with 100 kDa nominalmolecular weight limit Ultracel YM-100 regenerated cellulose membranewas identified as the product of choice. Centrifuge filter devices witha polyethersulfone filtration membrane were not feasible for thisinvestigation due to significant non-specific binding of the CPNPs tothe filtration membrane.

The conjugation of biotinylated human holotransferrin or biotinylatedanti-CD71 antibody to Avidin-CPNPs permits targeting of the transferrinreceptor; the conjugation of biotinylated pentagastrin to Avidin-CPNPspermits targeting of the gastrin receptor (FIG. 1A). A 1 ml aliquot of3.2 mg/ml biotin conjugated human holotransferrin (Invitrogen Co.), 0.20ml of 1 mg/ml biotin conjugated anti-CD71 antibody (GeneTex Inc.), or0.50 ml of 1 mg/ml biotin conjugated pentagastrin (Bachem Co.) (in 0.1 NNH₄OH) was added to 1 ml of Avidin-CPNP complex. This reaction mixturewas stirred at 600 RPM for 60 minutes at room temperature. The resultingbiomolecule-Avidin-CPNP complex was then filtered to remove excessunconjugated biomolecule (human holotransferrin, anti-CD71 antibody, orpentagastrin). Human holotransferrin-Avidin-CPNPs andanti-CD71-Avidin-CPNPs were filtered by tangential flow diafiltrationusing a 500 kDa molecular weight cut-off (MWCO) MicroKros hollow fibertangential flow diafiltration device (Spectrum Laboratories Inc.).Pentagastrin-Avidin-CPNPs were centrifuge filtered with a 100 kDacentrifuge filter device (Amicon Ultra-4, PLHK Ultracel-PL Membrane;Millipore Co.). All the biomolecule-Avidin-CPNP samples were filteredthree times in order to maximize the removal of excess unconjugatedbiomolecule. After each filtration step, the final volume of theretentate solution was brought back to the starting volume by theaddition of PBS.

Maleimide Coupling:

The conjugation of gastrin-10 to CPNPs via the PEG-maleimide couplingstrategy (FIG. 1B) permits targeting of the gastrin receptor. A 9 mlaliquot of citrate-CPNPs was chemically conjugated with maleimidepolyethylene glycol amine (PEG maleimide; JenKem Technology Inc.),through the ethyl-N-(3-dimethylaminopropyl)-N′ hydrochloridecarbodiimide reaction (EDCI, Fluka BioChemika ≧99.0% (AT); Sigma-AldrichCo.). The sample was first stirred at 550 rpm on a combined magneticstir/hot plate set to 50° C. In a drop wise manner, 1 ml of EDCI (1mg/ml) followed by 1 ml of PEG-maleimide (10 mg/ml), both in aqueoussolutions of CO₂-free deionized H₂O (pH 7), were added to the sampleunder continuous stirring, to produce a calculated 6-fold excess formonolayer surface coverage. The particles were left to react for 15hours at 50° C. to form amide linkages between the carboxylate surfacesand the PEG-maleimide. The mixture was then centrifuge filtered througha 100 kDa filter (Amicon PLHK Ultra PL-4 Membrane; Millipore Co.) at5000 g for 2 minutes to remove any excess EDAC and unreactedPEG-maleimide. The characterization of the resulting maleimide-PEG-CPNPsin the retentate showed that the CPNPs remained well dispersed after thecentrifugation wash. The gastrin-10 peptide has a cysteine residue forcovalent attachment. Thus, the gastrin-10 was added at a 5:1 molarexcess to the maleimide-PEG-CPNPs. This solution was incubated overnightat 4° C., protected from light, to produce Gastrin-10-PEG-CPNPs.

Sulfo-NHS-PEG Coupling:

In brief, citrate-functionalized CPNPs were activated by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), and then reactedwith sulfo-NHS to form a high-yield, semi-stable intermediate.Centrifugation was used to remove EDAC while PEG, with both amine andcitrate functional groups, was reacted with the sulfo-NHSester-containing CPNPs. This synthetic process was repeated with thecitrate-PEG functional terminals of the PEGylated CPNPs to generatesulfo-NHS ester-containing PEGylated CPNPs, which after furthercentrifugation to remove EDAC, readily reacted with anti-CD117antibodies to form specifically targeted, anti-CD117-PEG-CPNPs.

Polyethylene glycol (PEG) was conjugated to the surface of the CPNSPsusing an amide linkage between the amine termination of the PEG and thecarboxyl group from the citrate on the surface of the CPSNPs. Theconjugation procedure was modified from Altinoglu et al 2008. Theaddition of the N-hydroxysulfosuccinimide (Sulfo-NHS) increased theefficiency of forming the amide bond.

250 μ1 of EDAC (2 mg/ml CO2 free DI water) is added to 3 ml of citrateterminated CPSNPs dispersed in 70/30 ethanol/water. After 5 minutes, 250μl of Sulfo-NHS (15 mg/ml CO2 free DI water) is added to the solution.After another 5 minutes, a 250 μl solution of methoxy-PEG-amine (15mg/ml) and 250 μl solution of carboxy-PEG-amine (2 mg/ml) are addedsimultaneously. The particles are reacted for 15 hours at 50 degrees C.while stirring.

In contrast to the PEG-maleimide bioconjugation scheme, the carboxyterminal group of the carboxy-PEG-amine was used to conjugate antibodiesto the PEG surface of the CPNPs. 250 μl EDAC (1 mg/ml) is added tocarboxy-PEGylated CPSNPs while stirring at room temperature (25 C).After 5 minutes, 250 μ1 Sulfo-NHS (1 mg/ml) is added to the solution.The antibody solutions are added after 5 more minutes. For theconjugation of anti-CD96, 25 μl of a 6.67 μM solution is diluted into225 μl CO² free water and added into the particle solution. For theconjugation of anti-CD117, 5 μ1 of a 1.3 μM solution is diluted into 245μl CO2 free water and added to the particle suspension.

Characterization of Nanoparticles:

Particle size distributions for the CPNPs were obtained through dynamiclight scattering (DLS) using a Malvern Nano-S Zetasizer. Transmissionelectron microscopy (TEM) was performed using a JEOL JEM 1200 EXIIinstrument on dried nanoparticles prepared on a carbon film grid with acopper backing. To verify that avidin was grafted on the CPNPs, zetapotential distributions were obtained using a Brookhaven ZetaPALS zetapotential analyzer. To quantify and test the bioactivity of avidingrafted on CPNPs, a fluorometric assay for avidin-biotin interactionbased on the displacement of the fluorescent probe2-Anilinonaphthalene-6-sulfonic acid (2,6-ANS) was utilized (Mock etal., 1985). The reaction scheme for the 2,6-ANS assay is illustrated inFIG. 3.

A 4.85 mg/ml solution of 2,6-ANS (Molecular Probes, Invitrogen Co.) wasprepared in 1 ml of deionized H₂O. A 24 μg/ml solution of biotin wasprepared in 10 ml of deionized H₂O to produce the biotin solution forthe assay. The 2,6-ANS solution, followed by the biotin solution, wastitrated in 0.50 μl aliquots into the avidin-CPNP solution to obtain asufficient number of data points. The reaction mixture was stirred forone minute after the addition of each aliquot to the avidin-CPNPsolution to allow enough time for a homogeneous reaction mixture and tomaintain consistency with respect to reaction time throughout theexperiment. The fluorescence spectra were recorded with a PTIfluorometer in which emitted radiation is collected at 90 degrees with aphotomultiplier tube (PMT) detector. The sample is excited by a xenonarc lamp whose illumination passes through a 5 nm bandwidth slit and amonochrometer to select the excitation wavelength. For the emissionscan, the excitation wavelength was set to 328 nm and the emissionwavelength range was set to 350-625 nm.

Cell Culture:

Human MDA-MB-231 breast cancer cells were cultured in DMEM supplementedwith 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution(Invitrogen, Carlsbad, Calif.). Human BxPC-3 pancreatic cancer cellswere cultured in RPMI-1640 supplemented with 10% FBS and 1%antibiotic/antimycotic solution. All cell cultures were maintained at37° C. and 5% C0₂. Cells were harvested by trypsin/EDTA detachment forsubculture or tumor engraftment.

Fluorescence Microscopy:

Cells were grown on cover slips under normal media conditions, and thenincubated with targeted or untargeted CPNPs followed by a mediaexchange. Cells were fixed in 2% (w/v) paraformaldehyde, and mountedonto glass slides. Cells were visualized using a Nikon Eclipse E400microscope through a 40× objective using a combination Nikon DAPI/FIT-Cfilter cube and recorded on a Nikon Coolpix 995 digital camera.

Flow Cytometry:

Cells were detached from tissue culture-ware, surface Fc receptorsblocked with appropriate IgG, incubated with specific antibodies(anti-human CD71-FITC or-PE, eBiosciences, San Diego, Calif.), and fixedin 2% (w/v) paraformaldehyde. Samples were analyzed on a BD Biosciences(San Jose, Calif.) LSR II Special Order flow cytometer in the Penn StateCollege of Medicine Flow Cytometry Core, utilizing appropriatecompensation controls. Data analysis was performed using BD BiosciencesFACS Diva software.

In Vivo Tumor Xenograft Breast Cancer:

All animal procedures were approved by the Pennsylvania State UniversityInstitutional Animal Care and Use Committee. To evaluate the breastcancer-targeted CPNPs in vivo, xenografted MDA-MB-231 human breastcancer cells injected subcutaneously into athymic nude mice were used.Four to six week old female athymic nude mice were purchased from Harlan(Indianapolis, Ind.). Subcutaneous breast cancer xenografts wereprepared as previously described. Briefly, 10⁷ MDA-MB-231 cells,prepared in 100 μliter of growth media, were engrafted by subcutaneousinjection. Once the tumors established (one week), CPNPs suspended insterile isotonic saline were injected into the tail veins of the mice,and routine images were taken over a 96 hour period. As controls, freeICG or ICG-loaded, PEG-CPNPs (non-targeted) were alternatively injected,these were diluted to ensure that equivalent ICG concentrations (asdetermined by absorption spectroscopy) were administered to each mouse.

In Vivo Orthotopic Tumor Pancreatic Cancer:

To evaluate the pancreatic cancer-targeted CPNPs in vivo, an in vivomodel of pancreatic cancer, BXPC-3 human pancreatic cancer cells, wereorthotopically injected into the pancreas of athymic nude mice.Orthotopic pancreatic cancer tumors were prepared as previouslydescribed³⁰. Briefly, mice were fully anesthetized with a mixture ofketamine-HCl (129 mg/kg) and xylazine (4 mg/kg) and injectedintramuscularly. A small incision was made in the left flank, theperitoneum was dissected and the pancreas exposed. Using a 27-gaugeneedle, 10⁶ BxPC-3 cells, prepared in 100 μl of Hank's balanced saltsolution, were injected into the pancreas. All xenografted or orthotopictumors were allowed to establish for one week prior to experimentation.

In Vivo Imaging:

CPNPs, or controls, were diluted into sterile isotonic NaCl and 100μliter was injected via tail vein into tumor-bearing mice. EquivalentICG concentrations were determined prior to injection via absorptionspectroscopy (2×10⁻⁶ M prior to dilution). Whole animal imaging wasperformed as previously described. [2] Briefly, anesthesia was inducedand maintained by inhalation of 5% IsolSol (Vedco, St. Joseph, Mo.) in100% oxygen. Near-infrared transillumination images (755 nm excitation,830 nm emission, 3 minute exposure) and corresponding X-ray images wereobtained with an In Vivo FX whole animal imaging station (CarestrearnHealth, Rochester, N.Y.). Signal distribution relative to anatomy wasillustrated by merging near-infrared and X-ray images.

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We claim:
 1. A resorbable non-agglomerated calcium phosphosilicate CPNP(CPNP) coupled to a targeting moiety specific to one or morebiomolecules.
 2. The CPNP of claim 1 further comprising a dye, aradioactive material, a magnetic material, a therapeutic agent or anycombination thereof incorporated into the bulk of the CPNP.
 3. The CPNPof claim 2 wherein the CPNP is coupled to a targeting moiety by anon-covalent bond.
 4. The CPNP of claim 3 wherein the CPNP is coupled tothe targeting moiety by an avidin-biotin linkage.
 5. The CPNP of claim 4of wherein the targeting moiety comprises an antibody, a peptide, aligand, a receptor, or any combination thereof.
 6. The CPNP of claim 5wherein the antibody comprises an anti-CD71 antibody.
 7. The CPNP ofclaim 5 wherein the peptide comprises a holotransferrin.
 8. The CPNP ofclaim 5 wherein the peptide comprises a gastrin.
 9. The CPNP of claim 8wherein the gastrin comprises pentagastrin.
 10. The CPNP of claim 2wherein the CPNP is coupled to a targeting moiety by a covalent bond.11. The CPNP of claim 10 wherein the CPNP is coupled to the targetingmoiety by a polyethylene glycol-maleimide linkage.
 12. The CPNP of claim11 wherein the targeting moiety comprises an antibody, a polypeptide, aligand, a receptor, or any combination thereof.
 13. The CPNP of claim 11wherein the polypeptide comprises a gastrin.
 14. The CPNP of claim 13wherein the gastrin comprises gastrin-10.
 15. A method of preparing aCPNP coupled to a targeting moiety comprising: a) reacting a calciumphosphosilicate CPNP having a carboxylate surface functionalization witha carbodiimide and avidin to yield an avidinylated calciumphosphosilicate CPNP; and b) reacting a biotinylated targeting moietywith the avidinylated CPNP.
 16. The method of claim 15 wherein thecarboxylate surface functionalization comprises a citrate.
 17. Themethod of claim 15 wherein the carbodiimide comprises1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.
 18. Themethod of claim 15 of wherein the biotinylated targeting moietycomprises an antibody, a polypeptide, a ligand, a receptor, or anycombination thereof.
 19. The method of claim 18 wherein the biotinylatedantibody comprises an anti-CD71 antibody.
 20. The method of claim 18wherein the biotinylated polypeptide comprises a holotransferrin. 21.The method of claim 18 wherein the polypeptide comprises a gastrin. 22.The method of claim 21 wherein the gastrin comprises pentagastrin. 23.The method of claim 15 further comprising incorporating into the CPNP afluorescent dye, a radioactive material, a magnetic material, or anycombination thereof.
 24. The method of claim 23 wherein the dyecomprises indocyanine green, cascade blue, Rhodamine WT, fluorescein, orany combination thereof.
 25. The method of claim 15 further comprisingincorporating into the CPNP a chemotherapeutic agent.
 26. A method ofpreparing a CPNP coupled to a targeting moiety comprising: a) reacting aCPNP comprising calcium phosphosilicate and citrate with a carbodiimideand a maleimide polyethylene glycol amine so as to give rise to a CPNPcomprising a surface moiety having a maleimide terminal group; and b)reacting a binding moiety having a terminal sulfhydryl group with theCPNP.
 27. The method of claim 26 wherein the carbodiimide comprisesethyl-N-(3-dimethylaminopropyl)-N′ hydrochloride carbodiimide.
 28. Themethod of claim 26 wherein the sulfydryl-terminated binding moietycomprises an antibody, a polypeptide, a ligand, a receptor, or anycombination thereof.
 29. The method of claim 34 wherein thesulfhydryl-terminated binding moiety comprises a polypeptide.
 30. Themethod of claim 29 wherein the polypeptide comprises gastrin-10.
 31. Themethod of claim 26 further comprising incorporating into the CPNP afluorescent dye, a radioactive material, a magnetic material, or anycombination thereof
 32. The method of claim 31 wherein the dye comprisesindocyanine green, cascade blue, Rhodamine WT, fluorescein, or anycombination thereof.
 33. The method of claim 26 further comprisingincorporating into the CPNP a chemotherapeutic agent.
 34. A method ofpreparing a CPNP coupled to a targeting moiety comprising: a) activatingcitrate-functionalized CPNPs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; b) reacting the CPNPs with sulfo-NHS(N-hydroxysuccinimide) to form a high-yield, semi-stable intermediate;c) reacting PEG, with both amine and citrate functional groups, with thesulfo-NHS ester-containing CPSNPs; d) repeating the process with thecitrate-PEG functional terminals of the PEGylated CPSNPs to generatesulfo-NHS ester-containing PEGylated CPSNPs; and e) reacting withtargeted antibodies, at neutral pH, to form specifically targeted,PEGylated CPSNPs.
 35. The method of claim 34 wherein the targetedantibody is anti-CD117.
 36. The method of claim 34 wherein the targetedantibody is anti-CD96.
 37. A method of treating a disease byadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising: a) bioconjugated CPNPs possessingtargeting molecules complementary to one or more biomolecules present ina subject exhibiting a disease; and b) a chemotherapeutic agent integralto the CPNPs.
 38. The method of claim 37 wherein one or more of thetargeting molecules comprises a polypeptide, an antibody, a ligand, areceptor, or any combination thereof bound to a polyethyleneglycol-maleimide molecule.
 39. The method of claim 37 wherein one ormore of the CPNPs traverses the blood-brain barrier of the subject. 40.The method of claim 37 wherein one or more of the targeting moleculescomprises a polypeptide, an antibody, a ligand, a receptor, or anycombination thereof bound to an avidin-biotin complex.
 41. A method ofassaying a subject for a disease by administering CPNPs comprising: a) atargeting molecule complementary to one or more biomolecules present ina subject exhibiting a disease state, and b) a material that willindicate its location within the subject.
 42. The method of claim 41wherein the material that indicates its location within the subject is afluorescent dye, a radioactive material, a magnetic material, or anycombination thereof.
 43. The method of claim 41 wherein the targetingmolecule comprises a polypeptide, an antibody, a ligand, a receptor, orany combination thereof bound to a polyethylene glycol-maleimidemolecule.
 44. The method of claim 43 wherein one or more of the CPNPstraverses the blood-brain barrier of the subject.
 45. The method ofclaim 41 wherein one or more of the targeting molecules comprises apolypeptide, an antibody, a ligand, a receptor, or any combinationthereof, bound to an avidin-biotin complex.